Proliferation and survival of tumor cells involves a complex interplay between cell adhesion and mitogenic signal transduction cascades. EL4, a murine thymoma cell line, is a unique model system in which to investigate the molecular steps involved in these events. EL4 cells exist in two basic phenotypes, PMA-sensitive and PMA-resistant. These phenotypes refer to the ability of the cells to survive and proliferate in the presence of phorbol 12-myristate 13-acetate (PMA), a tumor promoter. Characterization of signal transduction and protein expression in the two cell types has revealed that PMA-resistant cells are unable to fully activate the Erk mitogen-activated protein kinase cascade in response to PMA. Erk activation is required for PMA-induced growth arrest in PMA-sensitive cells. Additional findings are that only PMA-resistant cells express focal adhesion kinase (FAK), while PMA-sensitive cells express higher levels of Pyk2, a FAK-related kinase. In addition, PMA-sensitive cells express higher levels of RasGRP, a guanine nucleotide exchange factor for Ras, than do resistant cells. RasGRP is a phorbol ester receptor that induces activation of Ras when bound to PMA. The proposed work tests two hypotheses that have arisen from these previous observations. The first is that, in EL4 cells, phorbol ester-mediated ERK activation occurs via proteins that regulate activation of Ras. The second is that expression of FAK enhances attachment, survival, and tumorigenicity of EL4 thymoma cells. The Specific Aims are: 1) To examine the effects of PKC activation on proteins upstream of Ras in EL4 cells, 2) To determine the roles of RasGRP and Pyk2 in PMA-induced Erk activation, 3) To define the roles of adhesion proteins in PMA response in EL4 cells, and 4) To examine the role of FAK and PMA sensitivity in tumorigenesis of EL4 cells in vivo. The proposed work will utilize a series of clonal EL4 cell lines. The alterations in protein expression that occur in these cells mimic those occurring in some tumors. EL4 cells provide a particularly novel model in which to study the roles of adhesion proteins in signaling. The long-term goal of this work is to delineate major pathways for response to phorbol ester and diglycerides. The information gained will suggest new therapeutic approaches to inhibit tumor cell progression.